Perturbation of the Fe-O-2 bond by nearby residues in heme pocket: Observation of nu(Fe-O2) Raman bands for oxymyoglobin mutants
Perturbation of the Fe-O-2 bond by nearby residues in heme pocket: Observation of nu(Fe-O2) Raman bands for oxymyoglobin mutants
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DOI：
10.1021/ja9608297
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发表时间：
1996-08-21
影响因子：
15
通讯作者：
Kitagawa, T
中科院分区：
文献类型：
--
作者：
Hirota, S;Li, TS;Kitagawa, T
Elucidation of Fe-O2 and O2-protein interactions in oxygenated heme proteins has been the focus of a large number of structural and spectroscopic studies. X-ray crystallographic analysis on oxymyoglobin (O2Mb) suggested the presence of a hydrogen bond between the distal histidine (His-64) and bound O2; a proposal was later confirmed in neutron diffraction experiments. 1 Vibrational spectroscopy, including infrared (IR) absorption and resonance Raman (RR) scattering, has provided more detailed information on protein ligand interactions. 2a-c, e, g Yu and co-workers2a suggested that the Fe-CO stretching (νFe-CO) frequency depends on the Fe-CO bond angle and accordingly reflects steric hindrance from nearby residues. More recent work has indicated that the electrostatic field near bound CO exerts a greater influence on the polarization of CO and its νFe-CO frequency than does steric hindrance. 2d-g The Fe-O2 complexes of Mb and hemoglobin (Hb) mutants have been much less studied, compared with their CO adducts, because of autoxidation problems. Since the OO stretching mode (νOO) couples with internal modes of the trans ligand (histidine), 3 it is difficult to deduce the intrinsic νOO frequencies from the observed frequencies. The Fe-O2 stretching (νFe-O2) mode has been identified for several end-on type O2-bound heme proteins by RR spectroscopy, 4 and its observed frequency directly reflects the strength of the Fe-O2 bond. We report here the νFe-O2 Raman bands for His-64 f Leu (H64L), Leu-29 f Phe (L29F), and Leu-29 f Trp (L29W) mutants of sperm whale Mb and discuss the effects of these residues on the Fe-O2 vibration.Preparation of mutant Mbs has been described elsewhere. 2e The purified protein was dissolved in 50 mM Na-phosphate buffer, pH 7.4. Crystals of L29W MbO2 were grown in the P6 form using 2.2 to 2.6 M ammonium sulfate, 20 mM Tris-HCl, 1 mM EDTA. 5a Diffraction data were collected on a Rigaku R-axis IIC imaging plate. The number of unique reflections was 17 658, and the starting model for molecular replacement was L29F MbO2. 5c Constrained least-squares refinement was performed by X-PLOR. After nine cycles of refinement and solvent placement, the crystallographic R-factor converged to 15.8%, with a final resolution of 1.8 A and 77.5% completeness. The coordinates and structure factors are being submitted to the Brookhaven Protein Data Bank. In order to observe the νFe-O2 Raman band of unstable oxy species, we used the oxygenation system originally developed for studies of cytochrome c oxidase. 6 About 20 mL of CO-bound Mb (10 µM) was circulated through the system at a flow rate of 20 mL/min. Oxygen (16O2 or 18O2) was incorporated into the solution just before the quartz Raman cell (cross section
